RESUMO
Highly purified RNA polymerase B (II) from calf thymus catalyses the synthesis of dinucleoside tetraphosphates from ribonucleoside triphosphates in the absence of an oligonucleotide primer or additional protein factors. The reaction requires a DNA template and bivalent cations such as Mn2+ or Mg2+. It is strongly inhibited by heparin and high concentrations of alpha-amanitin but not by rifampicin. On a given template various dinucleoside tetraphosphates of different sequence are formed although the yield depends on the nature of the template.
Assuntos
Oligonucleotídeos/biossíntese , RNA Polimerase II/metabolismo , Amanitinas/farmacologia , Animais , Bovinos , Fosfatos de Dinucleosídeos , Heparina/farmacologia , RNA Polimerase II/antagonistas & inibidores , Rifampina/farmacologia , Especificidade por Substrato , Moldes Genéticos , Timo/enzimologiaRESUMO
RNA polymerase from the archaebacterium Sulfolobus acidocaldarius was chemically modified with AMP o-formylphenyl ester followed by reduction with borohydride. The modified protein catalyzes the labeling of its own largest subunit when incubated with [alpha-33P]UTP in the presence of poly[d(A-T)]. On cleaving of the labeled protein using cyanogen bromide, hydroxylamine or amino acid-specific endoproteinases for a very brief period, the pattern and size of the radioactive fragments formed are best explained by attachment of the label between Gly843 and Met895 of the largest subunit. In this region there exists a highly conserved sequence which is also found in other archaebacterial, eukaryotic and prokaryotic RNA polymerase. This suggests that the binding site for the initiating substrate of RNA polymerases has been conserved during evolution.
Assuntos
Archaea/enzimologia , Bactérias/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , Marcadores de Afinidade , Sequência de Aminoácidos , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Hidrólise , Cinética , Dados de Sequência Molecular , Especificidade da Espécie , Especificidade por SubstratoRESUMO
DNA-dependent RNA polymerase B (II) from wheat germ was modified by incubation with 4-[N-(beta-hydroxyethyl)-N-methyl]benzaldehyde esters of AMP, ADP or ATP, followed by reduction with NaBH4. Reaction of the modified enzyme with [alpha-32P]UTP in the presence of various DNA templates led to a highly selective affinity labelling of the subunit with Mr 140 000 by covalently linked ApU. Labelling was inhibited by 1 microgram/ml alpha-amanitin.
Assuntos
Marcadores de Afinidade/metabolismo , RNA Polimerase II/metabolismo , Amanitinas/farmacologia , Radioisótopos de Fósforo , Triticum , Uridina Trifosfato/metabolismoAssuntos
Quimera , Enzimas , Mosaicismo , Sequência de Aminoácidos , Fenômenos Químicos , Química , Hibridização Genética , Proteínas , Especificidade da EspécieRESUMO
The catalytic activity of adenosine kinase (EC 2.7.1.20) from yeast is very labile. Even incubation with thiols provokes a loss of two thirds of its enzymatic activity. Concomitantly, two SH-groups appear on the enzyme in addition to the single SH-group already present in the untreated enzyme, the latter being absolutely essential for activity. Treatment of adenosine kinase with thiols does not substantially affect the binding of the substrates adenosine and ATP-Mg2. The reactivity of the two newly formed SH-groups is diminished in the presence of ATP-Mg2, whereas adenosine has no influence. The opposite holds for the reactivity of the single SH-group essential for enzymatic activity. Complete reactivation of the enzymatic activity after incubation of adenosine kinase with thiols can be achieved by reoxidation of the enzyme in presence of high concentrations of adenosine. These observations suggest the notion that adenosine kinase contains an essential SH-group close to the adenosine-binding site and a disulfide bridge near to the binding site of ATP-Mg2, the latter being easily accesible to the reduction by thiols.
Assuntos
Adenosina Quinase/metabolismo , Fosfotransferases/metabolismo , Compostos de Sulfidrila/farmacologia , Cisteína/farmacologia , Ácido Ditionitrobenzoico/farmacologia , Ditiotreitol/farmacologia , Cinética , Mercaptoetanol/farmacologia , Saccharomyces cerevisiae/enzimologiaRESUMO
Functionally equivalent subunits of RNA polymerase from Micrococcus luteus and Escherichia coli differ from each other in many molecular and antigenic properties. In spite of these differences, subunit alpha from E. coli and subunit beta from M. luteus form a complex alpha2beta, when incubated together. This complex binds rifampicin tightly, which the isolated subunits do not. The hybrid complex is very similar in its properties to the complex alpha2beta formed only from E. coli or M. luteus subunits. Since the sub-assembly alpha2beta from E. coli is reported to be an obligatory intermediate in the assembly process of complete RNA polymerase, the newly described hybrid sub-assembly may function similarly as an intermediate in the formation of the hybrid form of RNA polymerase described earlier.
Assuntos
RNA Polimerases Dirigidas por DNA , Escherichia coli/enzimologia , Micrococcus/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , Substâncias Macromoleculares , Peso MolecularRESUMO
DNA-dependent RNA polymerase lacking subunit sigma was digested with matrix-bound chymotrypsin or trypsin in the presence of 0.4 M NaCl in the monomeric form or at low ionic strength in the oligomeric form. Sigma-containing polymerase was digested in the same way. The course of proteolysis was followed by polyacrylamide gel electrophoresis after dissociation of the enzyme with detergent into subunits and the fragments produced by the hydrolysis. The following results were obtained. (a) The large subunits beta and beta' are cleaved with a much higher rate in the monomeric than in the oligomeric polymerase. (b) Both large subunits are hydrolysed with the same rate. (c) Subunit alpha is hydrolysed almost with the same rate in the monomeric and oligomeric form of polymerase. (d) The same was found for subunit sigma. (e) These effects were independent of the substrate specificity of the protease used. (f) Subunit sigma is much more susceptible to chymotrypsin than to trypsin. (g) Subunit sigma protects the large subunits beta and beta' against tryptic cleavage. These results can be explained in terms of a tentative model for the topography of the protomer-protomer interactions in RNA polymerase. According to this model subunits beta and beta' contain two sites for isologous interactions of protomers. One site can be blocked by attachment of subunit sigma. Subunits alpha and sigma do not participate directly in the association.
Assuntos
Quimotripsina , RNA Polimerases Dirigidas por DNA , Escherichia coli/enzimologia , Tripsina , Acrilamidas , Sítios de Ligação , DNA , Eletroforese em Gel de Poliacrilamida , Hidrólise , Cinética , Substâncias Macromoleculares , Maleatos , Concentração Osmolar , Polímeros , Conformação Proteica , Cloreto de Sódio , Dodecilsulfato de SódioRESUMO
We studied the inhibition of tryptic digestion of the subassembly alpha 2 beta of Escherichia coli DNA-dependent RNA polymerase to investigate its interaction with RNA and rifampicin. Both agents decreased distinctly the cleavage of subunit beta in the subassembly as well as the degradation of the transiently formed polypeptides (Mr greater than 80000). Short RNAs with a chain length of approximately 35 nucleotides were most protective at a concentration of 1 mg/ml while long RNAs were less effective at the same concentration. DNA did not exert any observable protective effects. The association of RNA with alpha 2 beta was shown by chromatography on phosphocellulose, which separates alpha 2 beta bound to RNA from free alpha 2 beta. The association of alpha 2 beta with RNA was inhibited by rifampicin.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , RNA/metabolismo , Rifampina/metabolismo , Escherichia coli/enzimologia , Substâncias Macromoleculares , Conformação de Ácido Nucleico , Saccharomyces cerevisiae , Tripsina/metabolismoRESUMO
When the potent immunosuppressive sesquiterpene ovalicin is added to lymphocyte cultures one first observes a preferential inhibition of uridine incorporation into rRNA. The uptake of the nucleoside, its conversion into the triphosphate or the polymerizing activity itself are not affected. A longer period of incubation with the drug results in a marked decrease in the number of ribosomes, with a concomitant reduction of the rate of leucine incorporation into all cellular proteins. After extended periods of time, the incorporation of thymidine into DNA in stimulated lymphocytes as well as in SV49.1 lymphoma cells is inhibited by 1 nM ovalicin or less, although part of the incorporation seems to be resistant to the drug even at much higher concentrations. A similar affect is observed with 3T6 mouse fibroblasts or HeLa cells. Here, however, a much longer incubation with the drug is required. This observation explains the selective effect of ovalicin on lymphocytes observed in vivo.
Assuntos
Imunossupressores/farmacologia , Linfócitos/imunologia , Sesquiterpenos/farmacologia , Animais , Linhagem Celular , Cicloexanonas/farmacologia , Replicação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HeLa/metabolismo , Humanos , Cinética , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfoma , Camundongos , Camundongos Endogâmicos , RNA/biossíntese , Ribossomos/metabolismoRESUMO
A new class of rifamycin-resistant mutants of Escherichia coli was obtained by lysogenic insertions of bacteriophage Mu Amp DNA. Rifamycin resistance is closely linked to the ampicillin resistance conferred by the prophage. Mapping by conjugation with auxotrophic markers revealed that the rifamycin-resistant mutations are located between 28 and 37 min on the E. coli chromosome standard map, some distance from the rpoB gene at 89.5 min. The DNA-dependent RNA polymerase of these mutants is highly sensitive to rifampicin.
Assuntos
Colífagos/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Genes Bacterianos , Genes , Mutação , Rifamicinas/farmacologia , Resistência Microbiana a Medicamentos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologiaRESUMO
RNA polymerase holoenzyme from Micrococcus luteus synthesizes in vitro a run-off transcript of 85 nucleotides from a DNA fragment containing part of gene E of bacteriophage phi X174. This RNA starts with GTP as the 5' terminus 18 nucleotides downstream from the start of gene E on the viral (+)strand. Transcription does not occur when the fragment is cleaved 36 nucleotides upstream of the initiation site. No transcript is obtained with RNA polymerase core or holoenzyme from Escherichia coli. Other DNA fragments containing the three major E. coli promoters of phi X174 are transcribed by both enzymes although much less efficiently by M. luteus RNA polymerase. When subunit sigma in E. coli RNA polymerase is replaced by sigma from M. luteus the resulting hybrid enzyme actively transcribes the DNA fragment containing the inner region of gene D with formation of the same run-off transcript which is obtained with M. luteus holoenzyme. In the presence of sigma from E. coli this RNA is not synthesized. The hybrid enzyme also transcribes a DNA fragment containing the gene A promoter of phi X174 with even higher efficiency than RNA polymerase holoenzyme from E. coli.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Óperon , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo , Bacillus subtilis/metabolismo , Sequência de Bases , Escherichia coli/metabolismo , Hidrólise , Micrococcus/metabolismo , Iniciação Traducional da Cadeia Peptídica , Especificidade da Espécie , Transcrição GênicaRESUMO
When extracts of Escherichia coli are filtered through a Sepharose column containing covalently bound rifamycin a protein is bound which can be eluted either with a high concentration of urea or more specifically with low concentrations of rifamycins. Its Mr is 18,000 +/- 1,000 in the presence of dodecylsulfate, in its absence 36,000 +/- 3,000. The association constant of the protein for rifampicin is 2.4 +/- 0.5 x 10(-4) M with two binding sites per dimer as determined by equilibrium dialysis. Large amounts of this protein are released from the cells by an osmotic shock.
Assuntos
Escherichia coli/metabolismo , Rifamicinas/metabolismo , Sítios de Ligação , Cromatografia de Afinidade , Cinética , Peso MolecularRESUMO
Incubation of DNA-dependent RNA polymerase from Micrococcus luteus (gram-positive) with the plasmid pBR322 under transcription conditions in vitro leads to the formation of a rather short-chained RNA. This transcript is initiated at the same site on pBR322 as RNA I, a defined Escherichia coli RNA polymerase product. M. luteus RNA polymerase initiates transcription at the RNA I site much more efficiently than the E. coli enzyme, using either a plasmid preparation of pBR322 or an appropriate linear restriction fragment as template. In the latter case, cleavage of the restriction fragment 24 or 71 nucleotides (but not 91 nucleotides) upstream of the initiation site destroys template activity. By sequence analysis it was determined that the 3' terminus of the M. luteus RNA polymerase transcription product is identical with that known for RNA I. Moreover, in agreement with synthesis of RNA I by E. coli RNA polymerase in vitro, termination by the M. luteus enzyme is also a stutter process characterized by the same dependence on the available UTP concentration. These observations lead to the hypothesis that termination by eubacterial RNA polymerases might not be species-specific.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Micrococcus/genética , Plasmídeos , RNA Bacteriano/biossíntese , RNA/biossíntese , Sequência de Bases , Escherichia coli/enzimologia , Micrococcus/enzimologia , Moldes Genéticos , Transcrição GênicaRESUMO
The spatial neighbourhood of the active center of Q beta replicase can be selectively modified by the method of self-catalysed affinity labeling. In the template-directed, mainly intramolecular enzymatic catalysis, the product [32P]GpG becomes specifically attached to the beta subunit. Using limited digestion of the radioactively labeled polypeptide by cyanogen bromide or N-chlorosuccinimide, we have mapped the attachment site to the region of subunit beta between Trp93 and Met130. Under our reaction conditions, Lys95 is the amino acid most likely to be modified, suggesting that Lys95 lies near the nucleotide binding site in the active center.
Assuntos
Marcadores de Afinidade , Q beta Replicase/química , Sequência de Bases , Catálise , Cromatografia em Camada Fina , Brometo de Cianogênio/química , Eletroforese em Gel de Poliacrilamida , Guanosina Trifosfato/química , Dados de Sequência Molecular , Fotoquímica , Succinimidas/químicaRESUMO
Highly purified RNA polymerase B (II) from wheat germ catalyses the formation of dinucleoside tetraphosphates from ribonucleoside triphosphates in the absence of an oligonucleotide primer or additional protein factors. The reaction requires bivalent cations such as Mn2+ or Mg2+ and proceeds linearly for several hours. It is strongly inhibited by 1 microgram/ml alpha-amanitin or 2 micrograms/ml heparin. The reaction strictly depends on the addition of a specific linear or circular DNA template, such as the plasmid pSmaF or a DNA fragment containing the gene for nopaline dehydrogenase. Bacteriophage T7 D111 DNA has almost no template activity. The start sites for dinucleotide synthesis on the template are limited. With the DNA fragment containing the gene for nopaline dehydrogenase only pppApA and pppApU are synthesised substantially whereas pppUpU is formed only in trace amounts. No significant dinucleotide synthesis is observed with other ribonucleoside triphosphates either singly or in a combination of two. The various regions of the DNA fragment differ distinctly in template activity.
Assuntos
Iniciação Traducional da Cadeia Peptídica , RNA Polimerase II/metabolismo , Catálise , Clonagem Molecular , Fosfatos de Dinucleosídeos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Oligonucleotídeos/metabolismo , Oligorribonucleotídeos/biossíntese , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/biossíntese , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Ribonucleotídeos/metabolismo , Especificidade por Substrato , Fagos T/metabolismo , Moldes Genéticos , Transcrição Gênica , TriticumRESUMO
A highly selective affinity labeling procedure has been applied to map the active center of DNA primase from the yeast Saccharomyces cerevisiae. Enzyme molecules that have been modified by covalent attachment of benzaldehyde derivatives of adenine nucleotides are autocatalytically labeled by incubation with a radioactive ribonucleoside triphosphate. The affinity labeling of primase requires a template DNA, is not affected by DNase and RNase treatments, but is sensitive to proteinase K. Both the p58 and p48 subunits of yeast DNA primase appear to participate in the formation of the catalytic site of the enzyme, although UV-photocross-linking with [alpha-32P]ATP locates the ribonucleoside triphosphate binding site exclusively on the p48 polypeptide. The fixation of the radioactive product has been carried out also after the enzymatic reaction. Under this condition the RNA primers synthesized by the DNA polymerase-primase complex under uncoupled DNA synthesis conditions are linked to both DNA primase and DNA polymerase. When DNA synthesis is allowed to proceed first, the labeled RNA chains are fixed exclusively to the DNA polymerase polypeptide. These results, in accord with previous data, have been used to propose a model illustrating the interactions and the putative roles of the polypeptides of the DNA polymerase-primase complex.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Marcadores de Afinidade/metabolismo , RNA Nucleotidiltransferases/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , DNA Primase , Replicação do DNA , Modelos Moleculares , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
We describe a method for specifically labelling T7 RNA polymerase at (or near) the active site. Enzyme molecules that have been modified by covalent attachment of a benzaldehyde nucleotide derivative in the presence of template DNA are subsequently incubated with radioactively labelled nucleoside triphosphates. Labelling of the enzyme occurs as a result of the formation of the first phosphodiester bond. The labelling is template-directed and the expected specificity of initiation at individual T7 promoters is observed. The label has been localized to an 80 kd tryptic fragment that contains the carboxy-terminal portion of the enzyme.
Assuntos
DNA Polimerase Dirigida por DNA , Fagos T/enzimologia , Marcadores de Afinidade , Sítios de Ligação , Nucleotídeos de Guanina , Marcação por Isótopo/métodos , Radioisótopos de FósforoRESUMO
DNA-dependent RNA polymerase from Micrococcus luteus can be isolated from cell extracts after removal of an excess of nucleic acids by fractionation with ammonium sulfate, followed by two consecutive gel filtrations through agarose and chromatography on cellulose phospate. Either homogeneous holoenzyme or a mixture of core and holoenzyme is obtained in this way, as is indicated by electrophoresis in polyacrylamide gels in the absence of detergent, where core enzyme migrates ahead of holoenzyme. Homogeneous core enzyme can be isolated from holoenzyme by chromatography on DEAE-cellulose. Core enzyme contains the subunits alpha, beta and beta' previously described [U.I. Lill et al., (1975) Eur. J. Biochem. 52, 411-420] in a molar ratio of 2:1:1. Holoenzyme contains an additional subunit sigma of 80 000 molecular weight (molar subunit composition alpha2 betabeta' sigma) and two relatively small polypeptides (molecular weight 14 000 and 25 000, respectively). Subunit sigma may be isolated from holoenzyme by chromatography on DEAE-cellulose at pH 6.9 in the presence of low concentrations of glycerol. The behaviour of holoenzyme during sedimentation in a glycerol gradient at low ionic strength indicates its occurrence as a dimer of the alpha2betabeta'sigma-protomer, whereas the monomeric form is preferred by core enzyme. Holoenzyme is much more active than core enzyme in RNA synthesis on bacteriophage T4DNA as template. The activity of the latter is stimulated by isolated sigma. M. luteus sigma as well as holoenzyme enhances also the activity of core enzyme fro- Escherichia coli. The formation of a hybrid between micrococcal sigma and E. coli core polymerase is also suggested by the influence of sigma on the oligomerisation of the enzyme from E. coli.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Micrococcus/enzimologia , DNA Viral , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Cinética , Substâncias Macromoleculares , Peso Molecular , Transcrição GênicaRESUMO
RNA polymerases A, B, and C from yeast were modified by reaction with 4-formylphenyl-gamma-ester of ATP as priming nucleotide followed by reduction with NaBH4. Upon phosphodiester bond formation with [alpha-32P]UTP, only the second largest subunit, A135, B150, or C128, was labeled in a template-dependent reaction. This indicates that these polypeptide chains are functionally homologous. The product covalently bound to B150 subunit was found to consist of a mixture of ApU and a trinucleotide. Enzyme labeling exhibited the characteristic alpha-amanitin sensitivity reported for A and B RNA polymerases. Labeling of both large subunits of enzyme A and B but not of any of the smaller subunits was observed when the reduction step stabilizing the binding of the priming nucleotide was carried out after limited chain elongation. These results illustrate the conservative evolution of the active site of eukaryotic RNA polymerases.